1. Field of the Invention
The present invention is concerned with a novel protein that inhibits the activation of transcription factor NF-κB by various signals that are important in inflammatory and immune processes. More particularly the invention relates to a protein herein designated NAP (Nemo Associated Protein), its recombinant production and its use.
2. Description of the Related Art
The Tumor Necrosis Factor/Nerve Growth Factor (TNF/NGF) receptor superfamily is defined by structural homology between the extracellular domains of its members Bazan, (1993). In general, with the exception of two receptors, the p55 TNF receptor and Fas/APO1, the various members of this receptor family do not exhibit clear similarity of structure in their intracellular domains. Nevertheless, there is much similarity of function between the receptors, indicating that they share common signaling pathways. This is seen in the ability of several receptors of the TNF/NGF family to activate the transcription factor NF-κB, by means of a cytoplasmic activator protein, TNF Receptor Associated Factor 2 (TRAF2). TRAF2 exerts its activity by binding to the structurally-dissimilar intracellular domains of several of the receptors of the TNF/NGF family.
TRAF2 is a member of a recently described family of proteins called TRAF (TNF Receptor Associated Factor) that includes several proteins identified as, for example, TRAF1, TRAF2, TRAF3, and TRAF6.
All proteins belonging to the TRAF family share a high degree of amino acid identity in their C-terminal domains, while their N-terminal domains may be unrelated. The TRAF2 molecule contains a ring finger motif and two TFIIIA-like zinc finger motifs at its C-terminal area. The C-terminal half of the molecule includes a region known as the “TRAF domain” containing a potential leucine zipper region extending between amino acids 264-358 (called N-TRAF), and another part towards the carboxy end of the domain between amino acids 359-501 (called C-TRAF) which is responsible for TRAF-binding to the receptors and to other TRAF molecules to form homo- or heterodimers.
Activation of the transcription factor NF-κB is one manifestation of the signaling cascade initiated by some of the TNF/NGF receptors and mediated by TRAF2. NF-κB comprises members of a family of dimer-forming proteins with homology to the Rel oncogene which, in their dimeric form, act as transcription factors. These factors are ubiquitous and participate in regulation of the expression of multiple genes. Although initially identified as a factor that is constitutively present in B cells at the stage of IgK light chain expression, NF-κB is known primarily for its action as an inducible transcriptional activator. NF-κB has many different activities in the cell, most of which are rapidly induced in response to extracellular stimuli. The majority of the NF-κB-activating agents are inducers of immune defense, including components of viruses and bacteria, cytokines that regulate immune response, UV light and others. Accordingly, many of the genes regulated by NF-κB contribute to immune defense (Grilli et al., 1993).
One major feature of NF-κB-regulation is that this factor can exist in a cytoplasmic non-DNA binding form which can be induced to translocate to the nucleus, bind DNA and activate transcription. The switching between these two forms of NF-κB is regulated by I-κB—a family of proteins that contain repeats of a domain that was initially identified in the erythrocyte protein ankyrin (Gilmore et al. 1993). In the unstimulated form, the NF-κB dimer occurs in association with an I-κB molecule which causes retention of the dimer in the cytoplasm and prevents its interaction with the NF-κB-binding DNA sequence and subsequent activation of transcription. The dissociation of I-κB from the NF-κB dimer constitutes the critical step of its activation by many of its inducing agents (DiDonato et al., 1995).
One of the most potent inducing agents of NF-κB is the cytokine tumor necrosis factor (TNF). There are two different TNF receptors, the p55 and p75 receptors, the expression levels of which differ according to cell type (Vandenabeele et al., 1995). The p75 receptor responds preferentially to the cell-bound form of TNF (TNF is expressed both as a beta-transmembrane protein and as a soluble protein) while the p55 receptor responds equally to both forms of TNF (Grell et al., 1995). The intracellular domains of the two receptors are structurally unrelated and bind different cytoplasmic proteins. Nevertheless, at least some of the effects of TNF, including the cytocidal effect of TNF and the induction of NF-κB, can be induced by both receptors. This feature is cell specific. The p55 receptor is capable of inducing a cytocidal effect or activation of NF-κB in all cells that exhibit such effects in response to TNF. The p75-R can have such effects only in some cells. Others, although expressing the p75-R at high levels, show induction of the effects only in response to stimulation of the p55-R (Vandenabeele et al., 1995). Apart from the TNF receptors, various other receptors of the TNF/NGF receptor family: CD30 (McDonald et al., 1995 and Berberich et al., 1994), the lymphotoxin beta receptor and, in a few types of cells, Fas/APO1 (Rensing-Ehl et al. 1995) are also capable of inducing activation of NF-κB. The IL-1 type I receptor, which also effectively triggers NF-κB activation, shares most of the effects of the TNF receptors despite the fact that it has no structural similarity to them.
The activation of NF-κB upon triggering of these various receptors results from induced phosphorylation of its associated I-κB molecules. This phosphorylation tags I-κB for degradation, which most likely occurs in the proteasome. The nature of the kinase that phosphorylates I-κB, and its mechanism of activation upon receptor triggering is still unknown. However, in recent years, there have been some advantages in relation to the identity of three receptor-associated proteins that appear to take part in initiation of the above-mentioned phosphorylation. A protein called TRAF2, initially cloned by D. Goeddel and his colleagues (Rothe, M. et al. 1994) seems to play a central role in NF-κB-activation by the various receptors of the TNF/NGF family. The protein, which when expressed at high levels can by itself trigger NF-κB activation, binds to activated p75 TNF-R, lymphotoxin beta receptor (Mosialos et al. 1995), CD40 (Rothe et al., 1995) and CD-30 (unpublished data) and mediates the induction of NF-κB by them. TRAF2 does not bind to the p55 TNF receptor nor to Fas/APO1, however, it can bind to a p55 receptor-associated protein called TRADD and TRADD has the ability to bind to a Fas/APO1-associated protein called MORT1 (or FADD). Another receptor-interacting protein, called RIP (Stanger et al., 1995) is also capable of interacting with TRAF2 as well as with FAS/APO1, TRADD, the p55 TNF receptor and MORT-1. Thus, while RIP has been associated with cell cytotoxicity induction (cell death), its ability to interact with TRAF2 also implicates it in NF-κB activation and it also may serve in addition to augment the interaction between FAS/APO1, MORT-1, p55 TNF receptor and TRADD with TRAF2 in the pathway leading to NF-κB activation. These associations apparently allow the p55 TNF receptor and Fas/APO1 to trigger NF-κB activation (Hsu et al., 1995). A protein called RAP-2 (RIP associated protein-2), now known as NEMO, is disclosed in WO 99/47672.
The triggering of NF-κB activation by the IL-1 receptor occurs independently of TRAF2 and may involve a recently-cloned IL-1 receptor-associated protein-kinase called IRAK (Croston et al., 1995).
By what mechanism TRAF2 acts is not clear. Several cytoplasmic molecules that bind to TRAF2 have been identified. However, the mechanisms by which TRAF2, which by itself does not possess any enzymatic activity, triggers the phosphorylation of I-κB is still uncertain.
In addition, it is to be noted that TRAF2 also binds to the p55 (CD120a) and p75 (CD120b) TNF receptors, as well as to several other receptors of the TNF/NGF receptor family, either directly or indirectly via other adaptor proteins as noted above. TRAF2 is thus crucial for the activation of NF-κB (Wallach, 1996). However, TRAF3 actually inhibits activation of NF-κB by some receptors of the TNF/NGF family, whilst TRAF6 is required for induction of NF-κB by IL-1 (Cao et al. 1996).
It is now known that TNF and the FAS/APO1 ligand, for example, can have both beneficial and deleterious effects on cells. TNF, for example, contributes to the defense of the organism against tumors and infectious agents and contributes to recovery from injury by inducing the killing of tumor cells and virus-infected cells, augmenting the antibacterial activities of granulocytes, and thus, in these cases the TNF-induced cell killing is desirable. However, excess TNF can be deleterious and as such may play a major pathogenic role in a number of pathological states such as septic shock, anorexia, rheumatic diseases, inflammation and graft-vs-host reactions. In such cases the TNF-induced cell killing has a deleterious effect. The FAS/APO1 ligand, for example, also has both desirable and deleterious effects. Binding of this ligand to its receptor induces the killing of autoreactive T cells during maturation of T cells, i.e. the killing of T cells which recognize self-antigens, during their development, thereby preventing the occurrence of autoimmune diseases. Further, various malignant cells and HIV-infected cells carry the FAS/APO1 receptor on their surface and can thus be destroyed by activation of this receptor by its ligand or by antibodies specific thereto, and thereby activation of cell death (apoptosis) intracellular pathways mediated by this receptor. However, the FAS/APO1 receptor may mediate deleterious effects, for example, uncontrolled killing of tissue which is observed in certain diseases such as acute hepatitis that is accompanied by the destruction of liver cells.
NF-κB is known to control the expression of many immune- and inflammatory-response genes. Thus, in view of the fact that the TNF/NGF family of receptors can induce cell survival pathways (via NF-κB induction) on the one hand and can induce cell death pathways on the other hand, there apparently exists a fine balance, intracellularly between these two opposing pathways. For example, when it is desired to achieve maximal destruction of cancer cells or other infected or diseased cells, it would be desired to have TNF and/or the FAS/APO1 ligand inducing only the cell death pathway without inducing NF-κB. Conversely, when it is desired to protect cells such as in, for example, inflammation, graft-vs-host reactions, acute hepatitis, it would be desirable to block the cell killing induction of TNF and/or FAS/APO1 ligand and enhance, instead, their induction of NF-κB, which would in turn lead to the enhanced expression of many immune- and inflammatory-response genes. Likewise, in certain pathological circumstances it would be desirable to block the intracellular signaling pathways mediated by the p75 TNF receptor and the IL-1 receptor, while in others it would be desirable to enhance these intracellular pathways.
It is an object of this invention to provide clones, proteins, and other tools for the modulation and/or mediation of NF-kB effects, in particular clones comprising the NAP protein and the NAP protein itself.
Citation of any document herein is not intended as an admission that such document is pertinent prior art, or considered material to the patentability of any claim of the present application. Any statement as to content or a date of any document is based on the information available to applicant at the time of filing and does not constitute an admission as to the correctness of such a statement.